1. Field of the Invention
This invention relates to polypeptides from phytopathogenic fungi with the biological activity of an inosine monophosphate dehydrogenase, to nucleic acids encoding them, to the use of the polypeptides and nucleic acids for identifying modulators of the polypeptides, to methods of identifying such modulators, and to the use of these modulators as fungicides.
2. Description of the Related Art
Undesired fungal growth which leads every year to considerable damage in agriculture can be controlled by the use of fungicides. The demands made on fungicides have increased constantly with regard to their activity, costs and, above all, ecological soundness. There exists therefore a demand for new substances or classes of substances which can be developed into potent and ecologically sound new fungicides. In general, it is customary to search for such new lead structures in greenhouse tests. However, such tests require a high input of labour and a high financial input. The number of the substances which can be tested in the greenhouse is, accordingly, limited. An alternative to such tests is the use of what are known as high-throughput screening (HTS) methods. This involves testing a large number of individual substances with regard to their effect on cells, individual gene products or genes in an automated method. When certain substances are found to have an effect, they can be studied in conventional screening methods and, if appropriate, developed further.
Advantageous targets for fungicides are frequently searched for in essential biosynthetic pathways. Ideal fungicides are, moreover, those substances which inhibit gene products which have a decisive importance in the manifestation of the pathogenicity of a fungus.
Inosine monophosphate dehydrogenase (EC 1.1.1.205), hereinbelow abbreviated to IMP dehydrogenase or IMPDH, catalyses the rate-limiting and irreversible step of de novo GTP synthesis. Inosine 5′-monophosphate (IMP) is reacted with nicotinamide adenine dinucleotide (NAD+) by IMPDH to give NADH/H+ and XMP (FIG. 1). Inosine 5′-monophosphate is the end product of de novo purine biosynthesis and an essential intermediate in the abovementioned synthesis of adenine and guanine nucleotides. Thus, IMPDH is a key enzyme for providing GTP, which is of enormous importance for the cell. GTP is incorporated into RNA and DNA, acts as substrate for GTPases (for example in cellular signal transduction pathways, such as G-protein-coupled receptors), for guanylate cyclase (generation of the secondary messenger substance cGMP) and as cosubstrate in many enzymatic reactions, and plays a role in the post-transcriptional modification of mRNA (5′-cap structure). IMPDH has therefore been a target for some time in the search for antiviral, immunosuppressive, carcinostatic and antimicrobial active compounds.
In addition, there is evidence that IMPDH, in plants, plays yet another, completely different, role which is associated with the assimilation, the transport and the storage of nitrogen. Thus, evidence exists that IMPDH is involved in the formation of ureids and allantoin.
Polypeptides with the activity of an IMPDH have also already been identified in specific fungi. These include the IMPDH from the fungus Candida albicans, which is pathogenic for humans and whose sequence encompasses 521 amino acids. The matching sequence can be obtained for example via what is known as the “trembl” database (U85049, AF249293). The sequences from the filamentous fungi Ashbya gossypii with 522 amino acids (trembl, A94860 and EP 0 927 761 A2), Saccharomyces pombe with 524 AA (Z97211) and Saccharomyces cerevisiae with 524 amino acids (Z46729), which are frequently employed in fermentations, and from the unicellular fungus Pneumocystis carinii, which is pathogenic for humans, with 529 amino acids (AF196975) are likewise available. Interestingly, several genes which encode a polypeptide with the activity of an IMPDH have already been identified in yeast.
Mycophenolic acid, a known specific IMPDH inhibitor, is a known medicament for suppressing the immune response for example following organ transplants. The structure of an IMPDH together with the inhibitor mycophenolic acid has already been elucidated (Sintack et al. (1996) “Structure and Mechanism of Inosine Monophosphate Dehydrogenase in Complex with the Immunosuppressant Mycophenolic Acid”. Cell 86, 921), and work on the biochemical mechanism of the inhibition of IMPDH by mycophenolic acid exists likewise (Fleming et al. (1996) “Inhibition of IMPDH by Mycophenolic Acid: Dissection of Forward and Reverse Pathways Using Capillary Electrophoresis”. Biochemistry 35, 6990).
Mycophenolic acid is highly effective against mammalian IMPDH, but markedly less effective as antimicrobial active compound (Digits et al. (1999) “Species Specific Inhibition of Inosine 5′-Monophosphate Dehydrogenase by Mycophenolic Acid”. Biochemistry, 38 15388). Mycophenolic acid binds to the intermediate which originates when XMP is formed from IMP into the nicotinamide binding pocket of the NAD+ binding site. However, owing to differences of the polypeptides derived from different species, the sensitivity to a human IMPDH is 20 to 450 times greater than to a microbial IMPDH (in this context, see also Zhang et al. (1999) “Differential Signatures of Bacterial and Mammalian IMP Dehydrogenase Enzymes.” Current Medicinal Chemistry 6, 537).
Further IMPDH inhibitors which are known in the field of medical therapy are, for example, ribovirin, a guanosine analogue, which has antiviral properties, tiazofurin, a C nucleoside which forms an NAD-like tiazofurin adenine dinucleotide following phosphorylation of the 5′-hydroxyl group and which is used as carcinostatic, or mizoribine, which is employed for immunosuppression in transplantation medicine (Goldstein and Colby (1999) “IMP Dehydrogenase: Structural Aspects of Inhibitor Binding”. Current Medicinal Chemistry 6, 519). In contrast, mizoribine is virtually ineffective against the fungus C. albicans, which is pathogenic for humans, and can apparently not be used at all as active substance against candidiasis (Ishikawa (1999) “Mizoribine and Mycophenolate Mofetil”. Current Medicinal Chemistry 6, 575).
The IMPDH sequence which has been isolated from C. albicans shows marked sequence similarity with the IMPDH from S. cerevisiae and other ogranisms. The ORF (open reading frame) of the C. albicans gene is interrupted by a small intron (289 base pairs (bp)) with typical exon-intron borders (Kohler et al. (1997) Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid. J. Bacteriol. 179, 2331). The growth of C. albicans cells can be inhibited by 1 μg/ml mycophenolic acid, while those cells which have a plasmid with recombinant IMPDH are resistant to up to 40 μg/ml.
In the case of Pneumocystis carinii, it has been shown that mycophenolic acid can act as an inhibitor of the IMPDH from this organism (O'Gara et al. (1997) “IMP dehydrogenase from Pneumocystis carinii as a potential target”. Antimicrob. Agents Chemother. 41, 40), and the exploitation of IMPDH as sensitive target of this organism has been proposed. The Pneumocystis carinii IMPDH (amino acid sequence) shows homology with bacterial IMPDH (31 to 38%), protozoal IMPDH (48 to 59%), mammalian IMPDH (60 to 62%) and fungal IMPDH (62%). A concentration of 25 μM mycophenolic acid resulted in 50% inhibition of the activity of the recombinant IMPDH.
Earlier work has shown that, as described above while mycophenolic acid inhibits in vitro the growth of some fungi which are pathogenic for humans (for example C. albicans, Cryptococcus neoformans), the compound is ineffective against Candida guilliermondii, Candida krusei, Candida pseudotropicalis, Hansenula anomala and also ineffective against S. cerevisiae (Noto et al. (1969) “Biological properties of mycophenolic acid”. J. Antibiot. (Tokyo), 22, 165).
These results show that mammalian IMPDH can be inhibited effectively with the known inhibitor mycophenolic acid, while the effect of the inhibitor is poor in the case of microorganisms. In the case of the fungi which are pathogenic to humans and which have been tested, the effect seems to be variable. Some fungi such as, for example, S. cerevisiae, have been described as being not sensitive to inhibition by an IMPDH inhibitor, while an effect, albeit a fungistatic rather than a fungicidal effect, can be observed in other fungi which are pathogenic for humans. The suitability of IMPDH as target protein for fungicides or for use in the search of fungicidal active compounds is therefore doubtful. In particular, an at least fungistatic effect has only been shown as yet in a few fungi which are pathogenic for humans, while no nucleic acid or amino acid sequence of an IMPDH from phytopathogenic fungi has been available as yet, and no results exist on whether phytopathogenic fungi might react sensitively to an IMPDH inhibitor so that such inhibitors can be used as fungicides.